NK Stimulatory Ligands
Natural Killer (NK) cells are large granular bone marrow-derived lymphocytes that serve as an important component of innate immunity and can attack virally infected cells, transformed cells and tumor cells (Trinchieri, G. Biology of natural killer cells. Adv. Immunol. 47: 187-376 (1989), Diefenbach et al. Strategies for target cell recognition by natural killer cells. Immunol. Rev. 181: 170-184 (2001), Moretta et al. Activating receptors and coreceptors involved in human natural killer cell-mediated cytolysis. Annu. Rev. Immunol. 19: 197-223 (2001)). NK cells act as a “rapid force,” responding faster than T cells and B cells as they do not have to rearrange the T cell receptor or the immunoglobulin genes to create a highly diverse repertoire of specificities against an antigen. Instead, NK cells recognize target cells by employing “missing-self” recognition [Ljunggren et al. In search of the ‘missing self’: MHC molecules and NK cell recognition. Immunol. Today 11: 237-244 (1990)].
NK cell activation is modulated by the balance between NK cell inhibitory receptor activity and NK stimulatory/activating receptor activity. Inhibitory NK receptor families include KIRs [Wilson et al. Plasticity in the organization and sequences of human KIR/ILT gene families. Proc Natl Acad Sci USA 97:4778 (2000)] in humans; the Ly-49 lectin-like homodimers [Takei et al. Ly49 and CD94/NKG2: developmentally regulated expression and evolution. Immunol Rev 181:90 (2001), Yokoyama et al. A family of murine NK cell receptors specific for target cell MHC class I molecules. Semin Immunol 7:89 (1995)] expressed in mice; and CD94-NKG2 lectin-like receptors expressed in both humans and mice. NK cell inhibitory receptors also bind to MHC class I molecules, which are important cell surface markers found in almost all cells, and are important in distinguishing self from non-self. The binding to these self-MHC molecules results in profound inhibition of the NK cell, and thus forms a basis for “missing self” recognition wherein the absence of MHC I leads to NK activation [Raulet et al. Regulation of the natural killer cell receptor repertoire. Annu. Rev. Immunol. 19: 291-330 (2001)].
A wide variety of NK cell activating receptors have been found in NK cells. See, e.g., Bahram et al., Curr. Op. Immunol. 2005, 17:505-519. Generally, activating receptors have short cytoplasmic domains and thus associate with transmembrane signaling adaptor molecules to activate NK cell function. NK cell activating receptors include NKG2A, NKG2C, NKG2D and NKG2E. Other activating receptors include: Natural Cytotoxicity Receptors (NCRs: NKp30, NKp44, NKp46); CD16 (responsible for ADCC); CD244 (2B4, can also make inhibitory signals); toll-like receptors (TLR); CD161; CD226 (DNAM-1); and CD96.
The sequence of NKG2A/C/E are highly related to each other and to C-type lectins. NKG2A/C/E are type-2 transmembrane receptors that are present in the NK cell membrane as heterodimers with another protein (CD94) and bind to non-classical MHC class 1 molecules known as HLA-E (in humans) or Qa1 (in mice) (Braud et al. Functions of nonclassical MHC and non-MHC-encoded class I molecules. Curr. Opin. Immunol. 11: 100-108 (1999)).
NKG2D, in contrast, is a homodimeric C-type lectin-like protein that is expressed by all NK cells, subsets of NKT cells and subsets of gamma delta T cells [Bauer et al. Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science 285, 727-729 (1999), Diefenbach et al. Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages. Nat. Immunol. 1: 119-126 (2000), Jamieson et al. The role of the NKG2D immunoreceptor in immune cell activation and natural killing. Immunity 17: 19-29 (2002)]. After stimulation, NKG2D is also expressed by virtually all CD8+ T cells and macrophages in mice [Diefenbach et al. Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages. Nat. Immunol. 1: 119-126 (2000), Jamieson et al. The role of the NKG2D immunoreceptor in immune cell activation and natural killing. Immunity 17: 19-29 (2002)].
Several distinct ligands for NKG2D have been identified, most of which are poorly expressed in normal cells but can be upregulated in infected, transformed and/or stressed cells. NKG2D ligands in humans include MHC class 1-chain-related protein A (MICA) and MICB (Bauer—1999), UL-16-binding proteins (ULBP) [Cosman et al. ULBPs, novel MHC class I-related molecules, bind to CMV glycoprotein UL16 and stimulate NK cytotoxicity through the NKG2D receptor. Immunity 14: 123-133 (2001)] and RAET1 [Radosavljevic et al. A cluster of ten novel MHC class I related genes on human chromosome 6q24.2-q25.3. Genomics 79: 114-123 (2002)]. The ULBP and RAET1 families are encoded on the syntenic region on human chromosome 6.
Mouse NKG2D ligands include histocompatibility 60 (H60) (Malarkannan et al. The molecular and functional characterization of a dominant minor H antigen, H60. J. Immunol. 161: 3501-3509 (1998)), Mouse UL16-binding protein-like transcript 1 (Mult1) (Carayannopoulos et al. Cutting edge: murine UL16-binding protein-like transcript 1: a newly described transcript encoding a high-affinity ligand for murine NKG2D. J. Immunol. 169: 4079-4083 (2002), Diefenbach et al. A novel ligand for the NKG2D receptor activates NK cells and macrophages and induces tumor immunity. Eur. J. Immunol. 33: 381-391 (2003)) and the retinoic acid early transcript 1 (Rae1) and its five alleles with >98% amino acid identity known as Rae1α-Rae1ε (Nomura et al. Genomic structures and characterization of Rae1 family members encoding GPI-anchored cell surface proteins and expressed predominantly in embryonic mouse brain. J. Biochem. 120: 987-995 (1996), Zou et al. Isolation and characterization of retinoic acid-inducible cDNA clones in F9 cells: a novel cDNA family encodes cell surface proteins sharing partial homology with MHC class I molecules. J. Biochem. 119: 319-328 (1996)). The Rae1, H60 and Mult1 families are only 20-28% homologous with each other. Interestingly, and analogous to human ULBP and RAET1, all known ligands for mouse NKG2D map close to the telomeric region of mouse chromosome 10 [Diefenbach et al. Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages. Nat. Immunol. 1: 119-126 (2000); Malarkannan et al. The molecular and functional characterization of a dominant minor H antigen, H60. J. Immunol. 161: 3501-3509 (1998); Nomura et al. Genomic structures and characterization of Rae1 family members encoding GPI-anchored cell surface proteins and expressed predominantly in embryonic mouse brain. J. Biochem. 120: 987-995 (1996)].
Tumor cells have developed many strategies for escaping immune surveillance, one of the ways is to shed the NKG2DL such as MICA (Groh et al. Tumor-derived soluble MIC ligands impair expression of NKG2D and T-cell activation. Nature 419:734-8 (2002); Salih et al. Cutting edge: down-regulation of MICA on human tumors by proteolytic shedding. J Immunol 2002; 169:4098-102) or ULBP2. Shedding of these ligands reduces the NKG2DL surface levels and effect the susceptibility to cytolysis by NK cells.
FAS
Higher organisms have developed several mechanisms to ensure the rapid and selective elimination of unwanted cells in various biological processes such as development, maintenance of tissue homeostasis, and elimination of cancer cells. One method of programmed cell death involves the interaction of cell surface Fas/CD95 with its cognate ligand, FasL/CD95L (Houston et al. The Fas signaling pathway and its role in the pathogenesis of cancer. Curr Opin Pharmacol. 4(4):321-6 (2004)).
Structurally, Fas is a transmembrane cell surface receptor containing three cysteine-rich extracellular domains at the amino terminus, which are responsible for ligand binding, and an intracytoplasmic death domain (DD) of about 80 amino acids that is essential for transducing the apoptotic signal [Peter et al. The CD95 (APO-1/Fas) DISC and beyond. Cell Death Differ 10:26-35 (2003)]. Binding of FasL to Fas causes a higher-order aggregation of the receptor molecules and recruitment of the adaptor molecule Fas-associated death domain (FADD) via DD-DD interactions. FADD also has another domain called the death effector domain, which in turn recruits pro-caspase-8 (FLICE) and/or pro-caspase-10 to the receptor. The resulting multimeric protein complex is called the death-inducing signaling complex (DISC), and forms within seconds of receptor engagement [Peter-2003].
Tumor cells may use the Fas signaling pathway to evade the immune response. One common mechanism is to decrease sensitivity of tumor cell to Fas-mediated apoptosis by regulating cell surface expression of Fas [Moller et al. Expression of APO-1 (CD95), a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium. Int J Cancer 57:371-377 (1994); Ivanov et al. FAP-1 association with Fas (Apo-1) inhibits Fas expression on the cell surface. Mol Cell Biol 23:3623-3635 (2003)]. In this approach tumors cells escape killing by NK cells and other effector cells by failing to express FAS receptor. Alternate approaches for evading the immune response include the secretion of an antagonistic ‘decoy’ receptor [Pitti et al. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 396:699-703 (1998)]; expression of anti-apoptotic molecules such as BCL2 family members (Sarid et al. Kaposi's sarcoma-associated herpesvirus encodes a functional bc1-2 homologue. Nature Med. 3: 293-298 (1997); Boise et al. BCL-X, a BCL-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74: 597-608 (1993)); down regulation and mutation of pro-apoptotic genes like BAX, APAF1 and CD95 (Ionov et al. Mutational inactivation of the proapoptotic gene BAX confers selective advantage during tumor clonal evolution. Proc. Natl. Acad. Sci. USA 97: 10872-10877 (2000); Soengas et al. Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature 409: 207-211 (2001); Teitz et al. Caspase-8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN. Nature Med. 6: 529-535 (2000); Strand et al. Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells—a mechanism of immune evasion? Nature Med. 2: 1361-1366 (1996)); alterations of p53 pathway (Bunz et al. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J. Clin. Invest. 104: 263-269 (1999); Schmitt et al. INK4A/ARF mutations accelerate lymphomagenesis and promote chemoresistance by disabling p53. Genes Dev. 13: 2670-2677 (1999)) or alterations of p13KT/AKT pathway (Kauffmann-Zeh et al. Suppression of c-Myc-induced apoptosis by Ras signaling through PI(3)K and PKB. Nature 385: 544-548 (1997); Chang et al. Transformation of chicken cells by the gene encoding the catalytic subunit of PI 3-kinase. Science 276: 1848-1850 (1997)). Cancer cells may also express FasL to induce apoptosis in immune cells.
The present specification describes a new and novel way of combating cancer by combining NK stimulatory molecules (such as Mult1) and a death domain (such as found in Fas). The engagement of NK cells and/or other immune cells with tumor cells expressing the fusion protein not only sends an apoptotic signal to the tumor cells but also activates the NK cells through the NKG2D receptor so that not only the engaged tumor cells will be killed via Fas induced-mechanisms but also are lysed directly by the activated NK cells.